The overall objective of this proposal is to employ modern techniques of protein engineering to develop a new generation of non-immunogenic and pharmacologically optimized enzyme for chemotherapy of hepatic cancers and melanomas by systemic L-Arginine depletion. Enzymatic depletion of arginine using PEGylated bacterial arginine deiminase (ADI) has been found to be of significant clinical benefit in the treatment of hepatoceullar carcinomas (HCC), renal cell carcinomas and melanoms. However the therapeutic utility of bacterial ADI enzymes is severely compromised by its high immunogenicity. We propose to engineer human enzymes that exhibit optimal catalytic, physical and pharmacokinetic properties without eliciting adverse immune responses. Combinatorial structure guided saturation mutagenesis, together with high throughput screening for arginine deiminase will be employed to generate two candidate enzymes: (i) mutants of peptidyl arginine deiminase 4 that hydrolyze L-Arginine instead of peptidyl arginine with high activity and (ii) engineered human Arginase variants exhibiting >10-fold lower KM for L-Arginine in plasma. The cytotoxic effect of these enzymes on various human HCC cell lines will be evaluated. Novel approaches for the modification of the engineered enzymes to achieved long serum persistence are described and these will be evaluated in mice. Finally, optimal dosing to achieve sustained depletion will be determined and tumor reduction and survival following administration will be assessed in human HCC xenografts. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinomas (HCC) kill hundreds of thousands of people worldwide every year. These cancers are very aggressive, and very difficult to treat, making new treatments of the utmost importance. However, one new treatment approach has recently been found that promises to keep these killers at bay. Hepatocellular carcinomas have lost the ability to make the amino acid L-Arginine, one of the building blocks necessary for cell growth. Cancer cells cope with this by scavenging L-Arginine from their surroundings and continue with unchecked growth. Excitingly though, when these tumors are treated with a bacterial enzyme that breaks down L-Arginine, these cancers starve to death while normal tissue is unharmed. The downside is that the body's immune system violently reacts to foreign particles, often making the treatment as dangerous as the disease. Our goal is take human enzymes and "tweak" them slightly so that they will efficiently break down L-Arginine. Dangerous immune responses will be avoided because these enzymes will be recognized as normal human proteins. In this proposal we will continue the engineering of two human enzymes (Peptidylarginine Deiminase and Arginase) that have already led to significant improvements in therapeutic potential. Using standard protein engineering techniques we will make highly active, and stable enzymes that will enable cancer cells to be specifically eliminated, without the harmful side-effects from using bacterial proteins. We believe that the research outlined here can create safe, effective, therapeutic agents for patients with hepatocellular carcinomas, and give our afflicted loved ones a second chance at life.